The Yeast Within

Can the good guys of the fungus world help us keep the bad guys in their place?

Geneticist Gerald Fink has a thing for yeast. "I've worked on yeast since 1962," he says. "My younger daughter was born in 1966, so I've known the fungus longer than I've known my daughter."

The familiarity shows. He casually refers to these microscopic, single-celled members of the fungus family as "our guys" and states, without exaggeration, "I've seen millions of 'em. When people in the lab show me something unusual, I always tell them, 'Well, yeah, you might think you have something new, but trust me. . . .' " All the more reason to appreciate the recent turn of events in Fink's lab at the Whitehead Institute for Biomedical Research in Cambridge,

Massachusetts. Not only did this familiar organism give him a whopping surprise, but--quite by chance--it may have shed light on how to deal with its many obnoxious fungal relatives.

Fungi are everywhere in nature--in the water, in the soil, on rotting vegetation. And they love to make themselves at home in our bodies. They cause mucous-membrane infections, skin infections such as ringworm and athlete's foot, and a variety of persistent lung diseases. In the otherwise healthy, these problems can be a mild to serious nuisance, but in those with faltering immune systems--people with AIDS or TB, transplant recipients, cancer patients undergoing chemotherapy--fungal infections can turn into an unmitigated disaster. They can spread corrosively through the body and brain, causing profound disability and death.

Fungal diseases, however, were far from Fink's mind in the spring of 1991, when this story properly begins. As usual, his lab was working on Saccharomyces cerevisiae, commonly known as baker's or brewer's yeast--the familiar variety that makes bread rise and juice ferment into beer and wine. Geneticists love this yeast for quite another reason. For such a simple organism, the yeast's cells and genes are astonishingly like ours but much easier to study. (Actually, though we tend to think of fungi as irredeemably plantlike, some studies suggest that they're more closely related to animals and therefore to us. Such studies vindicate what yeast geneticists have said all along: at the molecular level, at least, their guys can be terrific models for humans.)

Fink was in the midst of experiments exploring how genes control the growth of his lab's yeast cells. "How do their genes turn on and off in response to signals from the outside?" he wanted to know. "What happens, for example, when yeasts are faced with an environment in which they don't have enough nutrients?"

Not having enough nutrients is what wild yeasts face all the time. In contrast to their laboratory cousins, which are fed like royalty so as to produce fat, fast-growing specimens for study, yeasts and other fungi trying to make a living on a grape or a piece of cheese--or, for that matter, on your skin or inside your body--don't have the luxury of regular, abundant meals. Fink was imitating nature by manipulating the diet of his yeast. His lab was dotted with plates of Saccharomyces feeding on a gelatinous culture medium containing varying amounts of nitrogen, an essential nutrient. A lot of nitrogen, and the yeast cells grew gorgeously. Too little, and they didn't grow at all. At least that had been the general rule.

Then graduate student Carlos Gimeno walked into Fink's office bearing surprising news: a batch of yeast cells was acting strangely. The cells were forming filaments that snaked over the plate like unruly lines of conga dancers. Furthermore, these filaments were invading the jelly, as though in search of something. Gimeno was puzzled. Almost all other fungi carry on this way, at least some of the time, but never their Saccharomyces.

Indeed, one reason baker's yeast makes such a great laboratory tool is that it's so reliable. It predictably replicates to form separate, spherical cells. Daughter cells bud from their mothers to form independent cells that in turn produce their own daughters, and so on, creating what looks like a microscopic convocation of jelly beans. But Gimeno's nitrogen- starved yeast cells weren't behaving true to form. They had become oblong and skinny, and when daughter cells budded from their mothers they didn't detach into discrete cells; instead they remained connected end to end, like a string of sausage links. No wonder Gimeno wanted to talk.

Fink allowed that the growth was unusual, but he wasn't about to get excited. "I thought it was a contaminant," he recalls. Contamination with wild fungi can be a real headache in a lab. Their ubiquitous spores float in the air. They can easily get into the ventilation system and drift onto a culture plate, ruining an experiment. Fink was certain that was the story with Gimeno's yeast: " 'These are aliens,' I told Carlos. 'I've seen it all--these can't be our guys.' "

But one of the beauties of working with yeast is that it's possible to keep track of each strain (Fink has some 20,000 stashed in the freezer) because each carries its own characteristic genetic mutations, which usually manifest themselves as particular nutritional requirements. Upon checking, it became clear to the researchers that this was no contamination. The yeast cells were the very ones Gimeno had started with-- they were simply acting screwy.

Fink and Gimeno were thus poised at a delicious scientific crossroads: a confrontation with the unknown. "It's not like we do our experiments and wait at a machine, watching predictable data come out," says Fink. "We're always standing at the edge of a cliff, not knowing what to expect. Of course, that's where the fun lies--when we see something we haven't seen before. But the flip side is, the result could be nonsense. We have to convince ourselves that it's real."

Repeating the outcome would go a long way toward providing that assurance. So Gimeno started over, putting more of the same yeast strain on more of the same nitrogen-poor culture medium. Once again the yeast began its long, sinewy pilgrimage across, and into, the plate of jelly. Whatever was happening was legit.

But why was it happening? At a loss for an answer, Fink and his colleagues came up with a supposition. Normally lab yeasts are given all the nutrients they need. Well fed and content, they do what comes naturally in such a cushy situation: they replicate as fast as they can. But Gimeno's yeasts were starving. In response, they too did what comes naturally: they foraged for food. Since yeast cells are immobile, the only way they can forage is by transforming themselves into probing filaments. They slithered over and into the culture medium because they were just plain hungry.

The explanation made sense. So much sense, in fact, that Fink began wondering why they hadn't seen their baker's yeast behave like this before. "And how come we were seeing it," he asks, "and other people hadn't?"

Actually, some people had. In 1886 a Dane by the name of Emil Christian Hansen published a paper containing lovely drawings of yeast cells doing precisely what Gimeno's cells were doing--forming filaments. Hansen, who was the first person to grow pure cultures of Saccharomyces, had simply copied what he saw in nature. Taxonomists, whose job it is to observe and classify organisms rather than to investigate them at the molecular level, were also aware that yeasts could make filaments. They too saw it happening in the wild. But in the insular world of yeast genetics, the phenomenon was either unknown or dismissed as a nuisance. The reason was simple: geneticists prefer to work with stable organisms, so the ability to make messy filaments had long been bred out of Saccharomyces lab strains. It was simply serendipity that Gimeno had happened on a strain that, under stress, reverted to the long-lost behavior of its wild ancestors.

What was well known (even to yeast geneticists) was that other kinds of fungi, including those that cause grief in humans, grow filaments like crazy. One of them is the common yeast Candida albicans. Like fungal mold on a juicy piece of fruit, Candida sticks to moist body tissues. In infants it causes diaper rash and thrush, a common mouth infection, and in women vaginal infections. These garden-variety yeast infections are usually relatively easy to treat, but the same can't be said of all Candida invasions. Almost 1 in 14 hospital-acquired infections is caused by Candida--the fungus can slip into patients on inserted objects such as IV lines and catheters--and these infections can be dismayingly tenacious.

But it's people with poor immune defenses that Candida and other fungi hit hardest. In patients who have AIDS, or who are undergoing cancer chemotherapy, a local infection can spread unchecked throughout the body, lodging in organs such as the brain, heart, gut, kidneys, and eyes. The results are devastating: the fungus can bore into organs' cell surfaces, causing stubborn abscesses, progressive disability, and often death.

Systemic fungal infections like these are frustratingly hard to treat. "Fungi cling to cells. They are more difficult to eradicate than bacteria," says Harvard immunologist Alan Ezekowitz. And the very trait that makes fungi attractive to geneticists--their similarity to human cells--only makes things worse. For example, many antibiotics can attack features unique to bacteria without causing side effects in their human hosts. But drugs that destroy the vital processes of fungi tend to do the same to our own cells. "Most antifungal drugs work by destroying the fungal cell membrane or disrupting cell metabolism," says Ezekowitz. "But to some degree they also destroy human cells. That's why they cause toxicity--side effects such as muscle and joint pain, a drop in white blood cells, even kidney failure." The longer the treatment, the more likely the patient will experience such effects. The result can be self-defeating. "Fungal diseases are persistent and indolent," says Ezekowitz. "They require long-term therapy, but therapy is often limited by accumulated toxicity. There is a very real need for more specific antifungal drugs."

So far, though, the prospects have looked dim because Candida and other disease-causing fungi are simply not well understood. "People have been studying this organism for 40, 50 years, but there's an embarrassing paucity of information about what makes Candida pathogenic," says Fink. "The problem is, Candida is not a good lab organism. It's very difficult to manipulate genetically."

Fink therefore found it very intriguing that both Candida and his exotic lab yeast formed filaments. Not only that, but Candida, like his yeast, was known to switch from the round to the filamentous form when starved of nitrogen. Could it be that his docile baker's yeast might shed some light on pathogenic, intractable Candida? He and his colleagues are beginning to find out.

One of the fundamental questions they're trying to answer is, What role do filaments play in causing disease? In some Candida infections the affected tissue has only round fungal forms; in others it has filamentous forms; and in others yet, it has both. Ezekowitz thinks it's likely that both forms can bring about disease, although with different degrees of severity. Based on the insights offered by Fink's experiments, he offers a possible scenario. "Candida usually adheres to accessible body surfaces, such as the skin and mucous membranes," he explains. "If the conditions are optimal for it to survive--the right balance of acidity and alkalinity, and the appropriate nutrients--it just hangs around at the site."

If, on the other hand, the site is not congenial, the fungus may shift into its filamentous phase and take off for greener pastures, thereby spreading the infection. And, just as in Fink's culture plates, the filaments may penetrate the surface beneath them, gaining access to the bloodstream and, eventually, to internal organs. "The filamentous phase in Candida may be a response to a stressful situation--the fungus is trying to get away," says Ezekowitz. "It's an analogue to what happened in Fink's lab."

Fink, too, finds the scenario appealing. "Of course it's all pure conjecture," he says, "but at least it's now testable." Since Candida is so hard to work with, however, his lab is using baker's yeast as a means to understanding its tricky pathogenic cousin.

What the researchers have discovered so far is that in Saccharomyces, filament forming is controlled by a family of genes called BUD, whose role wasn't fully appreciated before. These genes do their job in a simple, straightforward manner: they direct where budding will occur on the yeast cells. For example, if you're a fat, well-fed yeast cell, it doesn't much matter where on your surface a daughter cell buds; the idea, after all, is to stay put. But if you're starving and must forage for food, then each daughter cell must bud her daughter in the same direction, and so on, until the resulting filament finds what it's looking for. The BUD genes, Fink found, are the architects of this outward-bound strategy.

"That budding pattern is absolutely critical to making filaments," says Fink. "If you're going to have directionality, all the buds have to come out in one direction--that's what the genes are for. But do equivalent genes control the appearance of filaments in Candida? And are they a factor in Candida pathogenicity?"

Fink and his colleagues are well on the way to answering the first question. One of the convenient traits of Saccharomyces, explains Fink, is that you can easily persuade it to take up foreign genes. "So what we've done," he says, "is take genes from Candida, shove them into Saccharomyces, and watch the outcome: Do any of these genes cause this shift?" The strategy has led Fink to a group of Candida genes--which his lab is now analyzing--that do appear to initiate filaments. "Knowing these genes are a good bet," says Fink, "we can now go back into Candida with what we've found and ask the second question: Do they play a role in Candida's disastrous spread?"

That's a far more difficult question, however. To answer it, Fink and his colleagues will have to take a Candida strain isolated from a patient and use genetic engineering to knock out "filament forming" genes in half the fungal samples. Then they'll take two groups of lab mice, inject one with the original fungus and the other with their new knockout fungus, and compare how the mice respond.

"You might find that there is no difference between the two groups," says Fink. "Then you would conclude that making filaments plays no role in how Candida causes disease. However, if you find that the guy who can make the filaments makes the mouse croak, while the mouse containing the one who can't make filaments is happy as a clam--it must mean that filaments have some role in pathogenesis. That experiment has never been done."

Fink and his colleagues are now doing it. If his hunch is right, he may have found an entirely new strategy against Candida and perhaps other fungal diseases as well. Rather than trying to kill off the hardy fungi completely, and almost killing the patient in the process, maybe it's only necessary to contain them. If you could make a drug that stops fungi from making filaments and spreading deeper into the body, you could keep the infection local and treatable--and this would mean new hope for curtailing the burgeoning problem of fungal disease. All this could come as a result of the strange behavior of a bunch of Fink's guys.